Evidence suggests that hypocretins (Hcrt), also called orexins, promote behavioral arousal and suppress rapid eye movement (REM) sleep. Loss or dysfunction of Hcrt system is associated with symptoms of narcolepsy including excessive sleepiness. In the brain, Hcrt neurons are localized within the perifornical-lateral hypothalamic area (PF-LHA). A majority of PF-LHA neurons are active during behavioral arousal and exhibit lower activities during quiet waking (QW) and non-REM sleep. In contrast, a majority of neurons in the ventrolateral preoptic area (VLPO) and the median preoptic nucleus (MnPN) of the preoptic region are active during sleep. Most of the sleep-active neurons in MnPN are GABAergic. MnPN GABAergic neurons constitute a source of afferents to PF-LHA. The identity of neurotransmitters/neuromodulators that regulate the state-dependent activity PF-LHA arousal systems is unknown. The role of Hcrts in regulating the state-dependent activity of sleep-active neurons in MnPN and VLPO is uncharacterized. First, we hypothesize that increased adenosinergic inhibition contributes to the suppression of PF-LHA neuronal activity during QW, and their further suppression during non-REM sleep results from increased gamma-amino butyric acid (GABA)-mediated inhibition. We will quantify the effects of adenosine and GABA agonists and antagonists delivered through an adjacent microdialysis probe on the state-dependent extracellular activity of PF-LHA neurons in freely behaving rats. Second, we hypothesize that the MnPN promotes sleep in part by inhibiting the wake-promoting systems in PF-LHA and that this inhibition is mediated via a GABAergic mechanism. We will examine effects of the MnPN electrical and chemical stimulation on the state-dependent activity of PF-LHA neurons in the presence and the absence of GABA receptor antagonist. The effects of GABAergic and adenosinergic drugs as well as effects of MnPN stimulation on Hcrt neurons will be further studied by double label immunostaining for hcrt-1 peptide and for c-Fos protein. We found that hcrt-1 microinjection into preoptic area promotes arousal. Third, we hypothesize that Hcrt system promotes arousal in part by inhibiting sleep-active neurons in MnPN and VLPO. We will determine the ability of focal microdialysis perfusion of Hcrts to suppress the activity of sleep-active neurons in VLPO and MnPN. Proposed studies will significantly advance our understanding of the neurotransmitter basis of the state-related control of PF-LHA arousal systems including Hcrt neurons and their interactions with preoptic sleep-promoting systems. This will contribute to the search for more targeted and specific therapeutic strategies for sleep disorders.